Original Article
The involvement of Nox4 in fine particulate matter exposure-induced cardiac injury in mice
2018 Volume 43 Issue 3 Pages 171-181
Details
2018 Volume 43 Issue 3 Pages 171-181
Epidemiological studies have confirmed that ambient fine particulate matter (PM2.5) exposure is associated with cardiovascular disease (CVD). However, the underlying mechanisms in PM2.5 exposure-induced heart injury are largely unknown. It has been acknowledged that NADPH oxidase (Nox) 4 plays a critical role in CVD development. To investigate the acute effects of PM2.5 on the mouse heart and the role of Nox4 in PM2.5 exposure-induced cardiac injury, C57BL/6J mice were instilled with saline or 1.5, 3.0, 6.0 mg/kg BW PM2.5 suspension for two weeks (five days per week). The levels of malondialdehyde (MDA), super oxide dismutase (SOD), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β in heart supernatants were determined using related kits. The expression of Nox4, p67phox, p47phox and p22phox in heart tissue was evaluated by immunofluorescence staining or Western blotting, respectively. Protein levels of p53, Bax, Bcl-2 and Caspase-3 in the heart were examined using immunohistochemical staining and Western blotting. TUNEL assay was used to measure myocardial apoptosis. PM2.5 exposure leads to obvious cardiac injury. PM2.5 exposure increases MDA level and iNOS activity, and decreases activity of SOD in heart supernatants of mice. High levels of TNF-α and IL-1β in heart supernatants of mice with PM2.5 instillation were determined. Nox4 and Nox-associated subunits such as p67phox, p47phox and p22phox expression levels were increased in heart tissue of mice after PM2.5 exposure. Additionally, PM2.5 exposure causes myocardial apoptosis in the mouse heart. This study suggested that Nox4 is involved in PM2.5 exposure-induced cardiac injury in mice.
Fine particulate matter (PM2.5) with an aerodynamic diameter (AD) less than 2.5 μm is acknowledged as the main cause of Chinese haze occurrence (Guarnieri and Balmes, 2014; van Donkelaar et al., 2010). PM2.5 can penetrate deeply into the alveolar regions of the lung and even transmit through blood into the circulation, including the heart, which has been attributed to adverse cardiovascular effects of haze (Brook et al., 2010; Guarnieri and Balmes, 2014; van Donkelaar et al., 2010). Epidemiological studies have provided convincing evidence that long-term or short-term PM2.5 exposure is associated with cardiovascular disease (CVD), including arrhythmia, atherosclerosis, myocardial ischemia and even heart failure (HF) exacerbation (Brook et al., 2010; Shah et al., 2013; Su et al., 2015). Lu et al. (2015) found that a 10 μg/m3 increase in PM2.5 relates to a 0.63% increase due to CVD mortality. Another study also reported that an increase of 10 μg/m3 in 2-day moving average concentration of PM2.5 corresponds to 1.22% increase of CVD mortality (Yang et al., 2011). The latest research (Shah et al., 2013) reviewed that per 10 μg/m3 increase in PM2.5 concentration is associated with 2.12% increase of heart failure (HF) hospitalisation or death.
Myocardial apoptosis plays a significant role in the pathogenesis of CVD (Cesselli et al., 2001; Dorn, 2009). Convincing evidence suggests that oxidative stress strongly induces cardiomyocyte apoptosis (Kaminski et al., 2002; Aikawa et al., 1997). Many animal experiments have suggested that PM2.5 exposure induces obviously oxidative stress in heart (Chen et al., 2017; Du et al., 2017). However, the underlying mechanisms in PM2.5-induced heart oxidative injury remain largely unknown. NADPH oxidases (Nox) are transmembrane enzymes, which are generally thought to be the main cause of oxidative stress by transferring an electron from NADPH to molecular oxygen (Qin et al., 2006). Oxidative stress products such as superoxide radical (O2-) and hydroxyl radical (-OH) can attack the cell membrane and further lead to tissue oxidative injury (Mak et al., 2002). Nox4, as the important Nox isoform expressed in the heart, has been acknowledged as the principal source of oxidative stress, which can also mediate myocardial apoptosis (Kuroda et al., 2010).
In the present study, we investigated the role of Nox4 in PM2.5-induced cardiac injury in mice to reveal the potential mechanisms underlying PM2.5-induced adverse cardiovascular effects, which could provide a useful strategy for the design of prevention measures against PM2.5 exposure-associated CVD.
The number of animals used and procedures employed were reviewed and approved by the Life Sciences Institutional Review Board of Zhengzhou University and performed strictly in accordance with the Guidelines of Zhengzhou University for Animal Experiments.
Animals6~8-week-old C57BL/6J mice (SPF degree, half male and female, 18-22 g in weight) were bought from the Medical Animal Center in Zhengzhou University, Henan, China, and raised in stainless steel cages in the Medical Animal Center located in the First Affiliated Hospital of Zhengzhou University (temperature 21-25°C, humidity 45%-55%, lights on 8 a.m.-8 p.m.).
PM2.5 sample collection and preparationThe sampling site was located in Zhengzhou during September, 2013. The quartz fiber filters (QFFs) (20.3 cm × 25.4 cm, Pall Corporation, New York, NY, USA) wrapped in aluminum foil were prebaked at 450°C for 4 hr and balanced at 25°C in desiccators. PM2.5 samples were collected on QFFs for 24 hr/d using a PM2.5 high-volume air sampler (Thermo, Anderson, SC, USA) with a pump flow rate of 1.15 m3/min. The QFFs were packed in aluminum foil and stored at -20°C until pretreatment after sampling. Chemical analyses of elements and polycyclic aromatic hydrocarbons (PAHs) of PM2.5 were performed as described in a previous study (Wang et al., 2014) and listed in Table 1 and Table 2 respectively. Particles were collected from filters by sequential sonications (four cycles of 20 min each) in sterile saline; detached particles were concentrated by vacuum freeze-drying and weighed. Saline was then added to the freeze-dried product to the concentration of 5.0 mg/mL. The size of particles was measured by transmission electron microscopy (TEM) (JEOL-2010F, Japan). The PM2.5 suspension was stored at 4°C and sonicated when it was instilled in mice.
| Elements | Concentrations (ng/m3) |
|---|---|
| Cu | 102.33 |
| Pb | 647.22 |
| Cr | 23.07 |
| Ni | 4.12 |
| Mn | 157.19 |
| Ba | 40.88 |
| Zn | 1598.07 |
| Al | 957.01 |
| As | 28.13 |
| Fe | 588.02 |
| Cd | 3.22 |
| Sb | 7.32 |
| PAHs | Concentrations (ng/m3) |
|---|---|
| Fluorene | 7.31 |
| Phenanthrene | 15.18 |
| Fluoranthene | 5.22 |
| Pyrene | 2.16 |
| Benzo (a) anthracene | 0.94 |
| Chrysene | 3.27 |
| Benzo (a) pyrene | 2.68 |
| Benzo (b) fluoranthene | 4.55 |
| Dibenzo (a,h) anthracene | 0.88 |
| Acenaphthylene | ND |
| Indeno (1,2,3-cd) pyrene | 1.99 |
| Benzo (k) fluoranthene | 4.02 |
| Benzo (g,h,i) perylene | 2.65 |
| Naphthalene | 0.12 |
| Acenaphthene | ND |
| Anthracene | ND |
The aim of this study is to disclose the role of Nox4 in short-term adverse effects on cardiovascular systems induced by PM2.5 exposure. Similar investigations have been previously based on very high PM exposure rates (Li et al., 2015; Cao et al., 2015). Starting from the dose proposed for repeated instillation protocol by Li et al. (2015), we reduced the cumulative dose of PM2.5 at 1.5, 3.0, and 6.0 mg/kg BW for two weeks in this study. Sixty mice were briefly exposed to isoflurane and kept under anaesthesia during the instillation procedure. Once a deep stage of anaesthesia was reached, mice were intratracheally instilled with 50 μL saline or 1.5, 3.0, 6.0 mg/kg BW PM2.5 suspension added to a total of 50 μL with saline for two weeks (five days per week) and were given free access to food and water during the non-performed time. Twenty-four hours after the last exposure, mice were euthanized with carbon dioxide.
Analysis of bronchoalveolar lavage fluid (BALF)The mice instilled with saline or PM2.5 were sacrificed and subjected to bronchoalveolar lavage. In detail, 1 mL of phosphate buffer saline (PBS) was instilled into the lungs of mice. The lavage fluids were collected and centrifuged at 1,500 rpm for 5 min at 4°C. The supernatants were collected for the following assays, while the cell pellets were resuspended in 0.2 mL of PBS balanced solution. Total cells were counted under an optical microscope. Level of total proteins in BALF was measured using BCA Protein Assay Kit (Tiangen Biotechnology, Beijing, China).
Measurement of malondialdehyde (MDA), super oxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) levels in mice heartsThe heart tissues from mice with saline or (1.5-6.0 mg/kg BW) PM2.5 instillation were weighed and homogenized in ice-cold PBS. After the homogenized solutions were centrifuged for 10 min at 3,000 rpm, the heart supernatants were harvested. The enzymatic activities of SOD, iNOS and MDA content in heart supernatants were determined by corresponding kits provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
Cytokines assay in heart supernatantsThe levels of TNF-α and IL-1β in heart supernatants from saline or (1.5-6.0 mg/kg BW) PM2.5-instilled mice were determined using ELISA kits (Cusabio Biotech, Wuhan, China).
Histological and pathologic examinationThe dissected mice lungs and hearts were fixed overnight in 4% paraformaldehyde at 4°C. After fixation, paraffin-embedded heart sections of 5 μm in thickness were stained with haematoxylin and eosin (H&E) or Masson’s Trichrome for light microscopy. To quantify the extent of cardiac fibrosis and hypertrophy, area of fibrosis (%) and cardiomyocyte area were determined. In detail, as described in published work (Weldy et al., 2013, Liu et al., 2010), area of fibrosis (%) was determined by Masson’s Trichrome stain where percentage of blue stain was quantified over total tissue area using Image-Pro Plus 6.0 software. Individual cardiomyocyte area was quantified using Image-Pro Plus 6.0 software, where the areas of 100 cardiomyocytes were quantified and averaged for each section. Of 100 cardiomyocytes, area was averaged to get a single value that represented average myocyte area per mice heart. We then averaged cardiomyocyte area within saline and PM2.5 exposure mice.
Immunofluorescence stainingThe slices of heart section were first blocked with goat serum for 30 min at room temperature, and then washed with PBS three times for 10 min. Next the slices were incubated with rabbit monoclonal antibodies specific for mouse Nox4 (Abcam, Cambridge, UK) with 1:500 dilution at 4°C for 30 min, followed by staining with Cy3 conjugated anti-rabbit secondary antibody (Beyotime Biotech Co. Ltd., Shanghai, China) with 1:200 dilution at 37°C for 60 min and protected from light. At last, DAPI was stained with 1:2,000 dilution at 4°C for 5 min. Images were captured using a Leica TCS-SP8 confocal microscope (Leica Microsystem, Wetzlar, Germany).
Western blottingHeart tissue proteins from mice exposed to saline and PM2.5, respectively, were extracted with a protein extraction kit (CWbio, Beijing, China) according to the manufacturer’s instructions strictly. The concentration of protein in the supernatants of tissue lysates was detected with a BCA Protein Assay Kit (Tiangen Biotechnology). Fifty μg of protein was mixed with 20 μL SDS loading buffer and boiled for 5 min. Then, 25 μg of protein from either saline or PM2.5 exposed mice was loaded on SDS-PAGE (10%-polyacrylamide) and subjected to electrophoresis. Separated proteins were transferred to PVDF membrane. Membranes were rinsed in 1 × TBST and blocked with 5% nonfat milk in TBST for 2 hr at room temperature. The rabbit monoclonal antibodies specific for mouse Nox4 (Abcam, 1:1,000 dilution), p53 (EnoGene, Nanjing, China, 1:500 dilution), Bax (EnoGene, 1:500 dilution), Bcl-2 (EnoGene, 1:500 dilution), Caspase-3 (EnoGene, 1:500 dilution), p67phox (Cell Signaling Technology, Danvers, MA, UK, 1:1,000 dilution), p47phox (Cell Signaling Technology, 1:1,000 dilution) and p22phox (Cell Signaling Technology, 1:1,000 dilution) diluted in 5% nonfat dry milk-TBST were incubated overnight at 4°C. Membranes were then washed 3 × 10 min with 1 × TBST, and incubated with HRP-conjugated secondary antibodies (CWbio) diluted in 1 × TBST to a final concentration of 1/3,000. After washing with 1 × TBST for three times, membranes were rinsed with ECL enhanced Chemiluminescence Kit (Vazyme Biotech, Nanjing, China). Images were developed using an Amersham Imager 600 system (Amersham Biosciences, Little Chalfont, Bucks, UK). The intensity of bands was quantified using Image J software.
Immunohistochemical (IHC)Expression of p53, Bax, Bcl-2, Caspase-3 in heart tissues from saline or (1.5-6.0 mg/kg BW) PM2.5-instilled mice was assessed by IHC. Briefly, heart sections were first blocked with goat serum for 30 min at room temperature, and incubated with rabbit anti-mouse p53 (EnoGene, 1:100 dilution), Bax (EnoGene, 1:200 dilution), Bcl-2 (EnoGene, 1:200 dilution) Caspase-3 (EnoGene, 1:150 dilution) monoclonal antibody over night at 4°C, followed by incubation with biotinylated goat anti-rabbit immnunoglobulin at a concentration of 1:100 at 37°C for 30 min. Positive IHC staining reflected as the brown staining in the cytoplasm was estimated by average optical density (AOD) in 10 high-power vision fields using Image-Pro Plus 6.0 software.
TUNEL assayMyocardial apoptosis in mice with saline or (1.5-6.0 mg/kg BW) PM2.5 instillation was measured using terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay kits provided by Nanjing Jiancheng Biotechnology Institute. Staining of tissue sections was performed according to the manufacturer’s protocol. Counterstaining with Mayer’s hematoxylin aided in morphologic evaluation in which normal nuclei were stained as blue and apoptotic nuclei as brown. Sections were viewed and photographed using optical microscopic techniques. The total number of TUNEL-positive cardiomyocytes in the heart was counted in three different fields for each section by an investigator who was blinded to the studies.
Data analysisAll experiments were repeated twice. Data are presented as mean ± standard error of the mean (S.E.M.). SPSS21.0 (IBM, Chicago, IL, USA) was used for statistical analysis. Data were compared by one-way analysis of variance followed by Dunnett’s post-test and two-tailed Student’s t-test.
As shown in Table 1, high levels of zinc (Zn), aluminum (Al), lead (Pb) and iron (Fe) were contained in the PM2.5 sample. In Table 2, PAHs was also detected and contained high levels of fluorine (FLU), phenanthrene (PHE), fluoranthene (FLT), benzo (k) fluoranthene (BkF), chrysene (CHR), benzo(a)pyrene (BaP) and benzo (b) fluoranthene (BbFA) in the PM2.5 sample. The size of particles instilled in this study is shown in Fig. S1, and was less than 2.5 μm.
PM2.5 exposure induces pronounced lung inflammation and damageMorphological and histological alterations in the lungs of mice instilled with saline or PM2.5 were evaluated by H&E staining. As shown in Fig. S2A, infiltration of inflammatory cells and injury of bronchial epithelium in lungs with PM2.5 exposure was observed. Total number of inflammatory cells and the level of total protein in BALF are markers of lung inflammation injury. Total number of inflammatory cells was counted using microscope and the level of total protein in BALF, indicator of lung epithelial damage, was measured using the BCA method. As shown in Fig. S2B, total number of inflammatory cells in BALF from mice instilled with PM2.5 suspension at the dose of 3.0 mg/kg BW was increased significantly compared to saline-treated mice (P < 0.05). The level of total protein in BALF (Fig. S2C) from PM2.5-instilled mice at the dose of 3.0 mg/kg BW was also significantly higher than that in saline controls (P < 0.05). These results indicated that PM2.5 exposure induces markedly inflammatory response and lung damage in mice, which may further cause systemic effects and then lead to cardiac effects.
PM2.5 exposure increases MDA level, iNOS activity and reduces SOD activity in mice heartsAs shown in Fig. 1, 3.0 mg/kg BW or 6.0 mg/kg BW PM2.5 instillation increased MDA level and iNOS activity in heart supernatants of mice compared to saline controls (P < 0.05). However, SOD activity was decreased in heart supernatants of mice instilled with 6.0 mg/kg BW PM2.5 compared to saline controls (P < 0.05). These results suggested that PM2.5 exposure induces obvious oxidative stress in heart.
PM2.5 exposure induces oxidative stress in mice hearts. Levels of MDA (A) and enzyme activity of SOD (B), iNOS (C) in heart supernatants from mice instilled with saline or 1.5-6.0 mg/kg BW PM2.5 were detected by corresponding kits provided by Nanjing Jiancheng Bioengineering Institute. Data are expressed as mean ± S.E.M. * P < 0.05, vs Saline (n = 7) and ** P < 0.01, vs Saline (n = 7).
After PM2.5 exposure, the hearts shape (Fig. S3A) of mice exposed to saline or PM2.5 is slightly changed. There was no significant statistical difference in ratio of heart weight and body weight (Fig. S3B) from mice with saline or PM2.5 exposure. Masson’s Trichrome and HE stained heart sections presented characteristic histological and morphological alterations. As shown in Fig. 2A, infiltration of inflammatory cells, large amounts of blue stain representing collagen fiber and phlegm, cardiomyocyte necrosis and hypertrophy in the hearts of PM2.5-instilled mice were observed, while the architecture of the hearts in saline-instilled mice was minimally affected. In order to define the extent of cardiac fibrosis and cardiomyocyte hypertrophy, area of fibrosis (%) and myocyte area (μm3) (Fig. 2B) were quantified by Image-Pro Plus 6.0 software. As a result, area of fibrosis (%) in cardiac tissues of mice instilled with 3.0 or 6.0 mg/kg BW PM2.5 was amplified in comparison with saline-treated mice (P < 0.05). Furthermore, myocyte area (μm3) in the hearts of mice with 3.0 or 6.0 mg/kg BW PM2.5 instillation were larger than that saline controls (P < 0.05). These results indicate that PM2.5 exposure leads to significant cardiac injury.
PM2.5 exposure induces heart pathological alterations in mice. Histological and morphological alterations (A) in hearts of mice with saline or 1.5-6.0 mg/kg BW PM2.5 instillation were observed by H&E staining and Masson’s Trichrome staining. Red arrows show infiltration of inflammatory cells in cardiac tissues from PM2.5-instilled mice. The yellow arrow shows cardiomyocyte necrosis in PM2.5-instilled mouse heart. Area of fibrosis (%) and myocyte area (μm3) (B) were quantified by Image-Pro Plus 6.0 software. Data are expressed as mean ± S.E.M. * P < 0.05, vs Saline (n = 5).
As shown in Fig. 3A, the level of TNF-α in hearts supernatant from mice instilled with 6.0 mg/kg BW PM2.5 was increased significantly compared to that observed in saline-instilled mice (P < 0.05). Moreover, higher level of IL-1β (Fig. 3B) in heart supernatant of mice with 3.0 mg/kg BW and 6.0 mg/kg BW PM2.5 instillation was determined (P < 0.05). These results suggested that PM2.5 exposure induces significant cardiac inflammation, which is consistent with a previous study (Li et al., 2015).
PM2.5 exposure increases levels of TNF-α and IL-1β in heart tissue. Levels of TNF-α (A) and IL-1β (B) in heart supernatants from mice instilled with saline or 1.5-6.0 mg/kg BW PM2.5 were determined using ELISA kits. Data are expressed as mean ± S.E.M. * P < 0.05, vs Saline (n = 7) and ** P < 0.01, vs Saline (n = 7).
As shown in Fig. 4A, the positive expression area of Nox4 in mice hearts is elevated with the increasing dose of PM2.5 exposure. Furthermore, the immunoblotting experiment showed that intensity of Nox4 bands in mice hearts with PM2.5 exposure (Fig. 4B) was also stronger than that in saline controls. Fig. 4C showed that PM2.5 exposure started to increase the expression of Nox4 in cardiac tissues at the dose of 3.0 mg/kg BW (P < 0.05). Expression levels of Nox-associated subunits p67phox, p47phox and p22phox are shown in Fig. 4D. Quantitative results of p67phox (Fig. 4E), p47phox (Fig. 4F) and p22phox (Fig. 4G) were also elevated after PM2.5 exposure. The results above suggested that PM2.5 exposure obviously up-regulates the Nox4 and Nox-associated subunits p67phox, p47phox and p22phox expression in mice hearts.
Effects of PM2.5 exposure on Nox4 and Nox-associated subunits expression in hearts tissue. The expression of Nox4 in mice hearts was determined using immunofluorescence staining (A) (bar = 50 μm) and immunoblotting (B). Relative expression of Nox4 (C) was quantified using Image J software. Expression levels of Nox-associated subunits p67phox, p47phox and p22phox were measured by Western blotting (D). Relative expression of p67phox (E), p47phox (F) and p22phox (G) was quantified using Image J software. Data are expressed as mean ± S.E.M. * P < 0.05, vs Saline (n = 3).
As shown in Fig. 5A, expression of p53, Bax, Bcl-2, Caspase-3 protein and TUNEL staining in fixed heart specimens prepared from saline or PM2.5-instilled mice was determined using IHC staining or TUNEL reagents. Western blotting analysis of p53, Bax, Bcl-2, Caspase-3 protein is shown in Fig. 5B. Our finding showed that PM2.5 exposure started to increase the expression of p53 (Fig. 5C) and Bax protein (Fig. 5D) in cardiac tissues at the dose of 3.0 mg/kg BW (P < 0.05) and decrease the expression of Bcl-2 (Fig. 5E) at the dose of 6.0 mg/kg BW (P < 0.05), while the ratio of Bcl-2/Bax (Fig. 5F) was increased. The expression level of Caspase-3 (Fig. 5G) in cardiac tissues from mice was increased at the dose of 6.0 mg/kg BW (P < 0.05). TUNEL experiment was used to detect myocardial apoptosis in the mouse heart. As shown in Fig. 5H, cells containing intensive TdT-positive staining (brown staining) in the nuclei were considered apoptotic. TUNEL positive nuclei (%) in cardiac tissues from (3.0 or 6.0 mg/kg BW) PM2.5-instilled mice was higher than that in saline-instilled mice (P < 0.05). These results suggested that PM2.5 exposure induces myocardial apoptosis in mice hearts.
Effects of PM2.5 on myocardial apoptosis in mice hearts. A: IHC staining of p53, Bax, Bcl-2, Caspase-3 and TUNEL staining in hearts from saline or PM2.5-instilled mice. B: Western blotting analysis of p53, Bax, Bcl-2, Caspase-3 in hearts from saline or PM2.5-instilled mice. Relative expression of p53 (C), Bax (D) Bcl-2 (E) and Caspase-3 (G) protein in cardiac tissues was evaluated by Image J software. F: Ratio of Bcl-2 and Bax. G: TUNEL positive cells in cardiac tissues from saline or PM2.5-instilled mice. Data are expressed as mean ± S.E.M. * P < 0.05, vs Saline (n = 3) and ** P < 0.01, vs Saline (n = 3).
It has been generally thought that ambient PM2.5 exposure is associated with adverse health effects such as respiratory disease and CVD causing Chinese haze (Brook et al., 2010; Guarnieri and Balmes, 2014). Several epidemiological studies showed that PM2.5 exposure relates to HF hospitalisations and HF mortality (Shah et al., 2013). The development of CVD results from various impairments, including cardiomyocyte apoptosis, cardiac remodeling and hypertrophy in cardiac function (George et al., 2014; Fujita and Ishikawa, 2011). It was also suggested that cardiac oxidative stress, inflammation and fibrosis played a vital role in the development of CVD (Butts et al., 2015; Weldy et al., 2013). However, the underlying mechanisms of PM2.5-induced adverse cardiovascular effects have not been well understood.
Ambient PM2.5 is a complex mixture of different chemical and microbial components, originating from different sources, which is suggested to be a major determinant of the adverse health effects (Jalava et al., 2015). In the present study, there are three options of cutter bars on the high-volume air sampler, which are the total suspended particulates (TSP), PM10 and PM2.5. Particulates with a diameter less than 2.5 μm can be collected on QFFs at the condition of PM2.5 cutter bar, while the other particulates (greater than 2.5 μm in diameter) were stopped and blocked by a special filter. TEM image of particles also suggested that the size of particles is less than 2.5 μm. Therefore, the particles we sampled were PM2.5. As known to all, the particles aggregate easily in sterile saline. For this reason, the PM2.5 suspension was sonicated to make particles dispersed just before intratracheal instillation into mice (Farina et al., 2011; Jin et al., 2017). We also determined that the chemical composition of PM2.5 samples was rich in some metals such as Zn, Al, Pb, Fe and part of PAHs. It has been reported that these components could deposit in terminal bronchioles, and then penetrate into the circulation causing injury in the heart (Guarnieri and Balmes, 2014; Brook et al., 2010). Published studies have reported that Zn compound exposure is associated with increased CVD mortality rate in the United States and PM-associated Zn relates to cardiac injury in rats (Chen et al., 2015; Kodavanti et al., 2008). It was also reported that Zn, Pb in PM2.5 can lead to heart rate and blood pressure increase (Cakmak et al., 2014). In addition, PAHs are also suggested to be associated with CVD (Baxter et al., 2014).
It has been reported that PM2.5 with a small size can penetrate deeply into the alveolar regions of the lung and even transmit through blood into the circulation, including the heart, and then triggers cardiovascular effects (Guarnieri and Balmes, 2014; Sancini et al., 2014). Our data showed that PM2.5 exposure results in lung inflammation and injury, which may further cause cardiovascular effects and then lead to cardiac injury. Convincing evidence from studies in vivo and in vitro suggested that PM2.5 can induce cardiac toxicity (Cao et al., 2016; Li et al., 2015). A cohort study indicated that long-term PM2.5 exposure associates with the increase of markers of inflammation (hs-CRP) in blood (Viehmann et al., 2015). In the present study, we found that PM2.5 exposure caused cardiac fibrosis and cardiomyocyte hypertrophy. However, there was no change in the ratio of heart weight/body weight. There are two reasons for this result. Firstly, the mouse heart is too small relative to body weight. After instillation of PM2.5, the instilled mice are not healthy like controls. Therefore, the weight loss of PM2.5-instilled mice reduces the difference of ratio among these four groups. Secondly, we weighed the hearts after washing with saline. For this reason, a small drop of saline may affect the results. All in all, PM2.5 exposure can directly and indirectly lead to heart injury through circulatory system. Next, we determined the underlying mechanisms of heart injury induced by PM2.5 exposure.
Oxidative stress is generally thought to be an important mechanism in determining the adverse effects of cardiorespiratory induced by PM2.5 (Weichenthal et al., 2013). PM2.5 can produce reactive oxygen species (ROS) such as superoxide radical (O2-) and hydroxyl radical (-OH) in vitro, and aqueous suspension of ambient PM2.5 may generate OH (Wu et al., 2012; Gehling et al., 2014). Under stress conditions, the activity of SOD an anti-oxidative enzyme may be depressed. Additionally, excessive ROS is easy to attack the cell membrane and form MDA, which is a typical product of lipid peroxidation (LPO). Also, excessive NO regulated by iNOS may form a peroxynitrite anion, which is more toxic than O2- and OH or other free radicals (Mak et al., 2002; Snyder and Bredt, 1992). In this study, we found that PM2.5 exposure increases MDA level and iNOS activity and reduces SOD activity in mice hearts, which is consistent with the above views. Taken together, these results indicate that PM2.5 can cause heart injury through generating oxidative stress products.
Oxidative stress has been considered an important mechanism of CVD development (Nabeebaccus et al., 2011; Brandes et al., 2010). Nox, a major source of oxidative stress in the cardiovascular system, is especially important in redox signaling. Accumulating in vitro and in vivo evidence suggested that Nox plays important roles in the pathogenesis of cardiac remodeling (Brandes et al., 2010; Heymes et al., 2003). It is known that Nox consists of a Nox catalytic subunit (Nox1-5, Duox-1 and Duox-2) and various regulatory molecules, such as p22phox, and p47phox. The prototypic Nox is composed of a membrane-bound heterodimer consisting of a catalytic Nox2 (gp91phox) subunit and a p22phox subunit and several cytosolic regulatory subunits: p47phox, p67phox, p40, and Rac (Murdoch et al., 2006). Among them, Nox4 is widely considered to be an emerging therapeutic target in CVD (Streeter et al., 2013). It has been reported that Nox4 plays a critical role in cardiomyocyte injury (Wang et al., 2017). In the present study, PM2.5 exposure caused cardiac inflammation, hypertrophy and fibrosis. Interestingly, increasing expression of Nox4 and Nox-associated subunits p67phox, p47phox and p22phox were detected in PM2.5-exposed hearts. A previous study indicated that Nox is involved in metal-rich particulate matter exposure-induced vascular dysfunction (Cuevas et al., 2015).
Apoptosis of cardiomyocytes is also considered to be an important contributor to the development of fibrosis and contractile dysfunction in the development of CVD (Dorn, 2009). It had been reported that apoptosis proteins such as p53, Bax, Bcl-2 and Caspase-3 play pivotal roles in myocardial apoptosis (Fujita and Ishikawa, 2011; Qin et al., 2006). p53 is one of the most famous proteins and a major tumor suppressor, which is recognized as a key molecule in the adaptation to a wide variety of harmful stimuli including oxidative stress(Fujita and Ishikawa, 2011). Pro-apoptotic protein Bax is a member of the Bcl-2 family, and mainly resides in the cytoplasm. It had been suggested that up-regulated Bax protein and down-regulated Bcl-2 protein activated the apoptotic pathway (Zhu et al., 2015; Gupta and Knowlton, 2005). In the cardiovascular system, p53 or Bax-mediated signaling pathway in the apoptosis of cardiomyocytes was recently shown to have a crucial function in the development of HF (Shizukuda et al., 2005; Qin et al., 2006). In our study, we found that PM2.5 exposure up-regulates p53, Bax and Caspase-1 protein in cardiac tissues and decreases the expression of Bcl-2, while the ratio of Bcl-2/Bax is also increased. Moreover, TUNEL assay was performed to examine myocardial apoptosis and the result showed that PM2.5 exposure induced myocardial apoptosis in the mouse heart. A published study (Wang et al., 2017) indicates that Nox4-mediated apoptosis signaling including p53 plays a critical role in cardiomyocyte injury. Taken together, these results suggest that PM2.5-induced cardiac injury may be associated with Nox4-mediated apoptosis signaling.
In conclusion, our study firstly found that Nox4 is involved in PM2.5 exposure-induced heart injury in mice. Of course, our research also has shortcomings. In the future, an inhibitor of Nox4 should be utilized to reveal the toxicological mechanisms of PM2.5-induced human CVD and provide positive information for prevention and treatment of PM2.5 toxicity.
The work was supported by the National Natural Science Foundation of China (No.81570274), by the research foundation of Science and Technology Department of Henan Province (No. 411045800, No. 094100510014).
Conflict of interestThe authors declare that there is no conflict of interest.
